Recent investigations into plant genes have focused on sequence-specific DNA-binding proteins that may play a role in trans regulation. These DNA-binding proteins are usually localized in the nuclei, and their target DNA sequences and binding specifications can be characterized by gel mobility-shift assays and DNAse footprinting techniques using either whole cell or nuclear extracts. Results from such in vitro experiments have led to the conclusion that 5' upstream regions of plant genes contain binding sites for multiple nuclear protein factors (cf. Allen et al., 1989, Plant Cell 1:623-631; Gilmartin et al., 1990, Plant Cell 2, 369-378; Schindler and Cashmore, 1990, EMBO J. 9:3415-3427). Moreover, in some cases, a single nuclear factor may interact with more than one promoter. For example, the tobacco nuclear factor, Activation Sequence Factor (ASF)-I, was first identified by its ability to bind to the TGACG motifs located in the -83 to -63 region of the Cauliflower Mosaic Virus (CaMV) 35S promoter (Lam et al., 1989). Further analyses, however, demonstrated that it also binds to a similar motif in the 5' regions of the wheat histone H3 gene (Katagiri et al., 1989, Nature 340:727-730), nopaline synthase gene (Katagiri et al., 1989, supra; 1989 Bouchez, et al., 1989, EMBO J. 8, 4197-4204; Lam et al., 1990, J. Biol. Chem 265:9909-9913), octopine synthase gene (Fromm, et al. 1989, Plant Cell 1:977-984; Tokuhisa et al., 1990, Plant Cell 2:215-224), and the TR1' and 2' promoters of octopine T-DNA (Bouchez et al., 1989, EMBO J 8:4197-4204). These biochemical results are consistent with previous genetic data indicating that a regulatory gene may control the activity of several structural genes (cf. Coe and Neuffer, 1977, In Sprague, G. F., ed., Corn and Corn Improvement. Madison, Wis., USA. pp. 111-223).
Another nuclear factor, the G-box binding factor (GBF), also appears to bind to several classes of promoters. Giuliano et al., (1988, Proc. Natl. Acad. Sci. USA 85:7089-7093) first reported that this factor binds to the G-box motif, 5'-TCTTACACGTGGCAYY-3' (SEQ. ID NO: 27) conserved in the upstream sequences of several dicotyledonous rbcS genes. A G-box related motif, containing the core sequence CACGTG is also present in the 5' regions of two other classes of light-responsive genes: the Arabidopsis cab genes (Ha and An, 1988, Proc. Natl. Acad. Sci. USA 85:8017-8021) and the chalcone synthase gene (chs) of Petroselium crispum (Schulze-Lefert et al., 1989, EMBO J.8:651-656; Schulze-Lefert et al., 1989, Plant Cell 1:707-714) and Antirrhinum majus (Staiger, et al., 1989, Proc. Natl. Acad. Sci. USA. 86:6930-6934) have demonstrated that the G-box motif in chalcone synthase is related to that of N. tabacum rbcS since the two sequences appear to compete for the same tobacco nuclear factor, CG-1. Their results suggest that CG-1 and GBF have similar binding specificities; however, it is not known whether the two protein factors are indeed identical or, just related. It should be noted that the G-box or related motif is not exclusively associated with light-responsive genes because it is also found at -577 of the patatin (PI-II) promoter (Rosahl et al., 1986, Mol. Gen. Genet. 203:214-220) and at -200 of the Arabidopsis alcohol dehydrogenase (Adh) promoter (McKendree et al., 1990, Plant Cell 2:207-214; DeLisle and Ferl, 1990, Plant Cell 2:547-557). These findings raise the possibility that GBF or CG-1 may simply be an ubiquitous factor capable of interacting with promoters of diverse regulatory properties.
Recently, a rice nuclear factor that binds specifically to a 5'-GTACGTGGCG-3' sequence (SEQ. ID NO: 28) of the rice rab16A promoter has been described (Mundy et al., 1990, Proc. Natl. Acad. Sci. USA 87,406-410). This sequence, designated as motif I, is conserved not only among all four ABA-responsive rab16 genes (A-D; Mundy and Chua, 1988, EMBO J. 7:2279-2286; Yamaguchi-Shinozaki et al., 1990, Plant Mol. Biol. 14:29-39), but also in cotton genes (lea) that are expressed during late embryogenesis (Baker et al., 1988, Plant Molecular Biology 11:277-291) as well as the Em gene of wheat (Marcotte, et al., 1989, Plant Cell 1:969-976). Comparison of motif I and the G-box motif reveals extensive sequence homology.